Poly(phosphoester) (PPE) as polymer backbone

Living organisms depend on different kinds of poly- and oligomeric phosphorus derivatives for storing genetic information (deoxyribonucleic acid/ ribonucleic acid (DNA/RNA)) or storing chemical energy (adenosine triphosphate (ATP)). The long-term stability of DNA derives from its negatively charged phosphate linkers, yet can be degraded via hydrolysis with enzymes.

The mimicking of those systems can achieve biocompatible and -degradable polymers with similar mechanical and chemical properties. The PPEs consist of pentavalent phosphorus, a backbone containing different kinds of aliphatic chains and the side chain, which can contain different functional groups, yielding poly(phosphate)s, poly(phosphonate)s and poly(phosphoamidate)s (Figure 14). PPEs are degradable by hydrolysis with or without enzymes, thus qualifying as biocompatible and -degradable. By changing the backbone or the side chain, the properties of the PPEs can be manipulated to generate amorphous, water-soluble materials or crystalline, stiff plastics.^[44]

18

Figure 14: Synthetic pathways toward PPE and fields of application.^[44]

Especially the biocompatible and -degradable properties of poly(phosphonate)s make them interesting materials for applications in drug delivery.^[49] In comparison to widely used biodegradable polymers like poly(lactic acid), poly(glycolic acid) and poly(ε-caprolactone), PPEs are degraded much faster by hydrolysis or enzymatic degradation. The polymer backbone and side chains of PPE degrade under basic conditions similarly fast, while the side chains also degrade under acidic conditions.^[50] The degradation rate can be adjusted by changing the chemical structure of the PPE.^[44] The degradation products of poly(phosphonate)s have been studied and show, that there is no toxic effect on COS-7 cells.^[51] However, further studies of poly(phosphonate) structures are important to prove general biocompatibility. If the backbone and the side chains are correctly chosen, the formation of hydrogels are possible. Cross-linking of a triblock copolymer of poly(ethyl ethylene phosphate) and poly(ethylene glycol) with a diacrylate yields nanogels as a drug carrier.^[52]

19 1.2.3 Dextran as polymer backbone

Dextran is a homopolysaccharide containing glucose monomer units with α-1,6 glycosidic linkage and a few branches of α-glucopyranose at the positions O-2, O-3 or O-4 (Figure 15).

Dextran is synthesized by lactic acid bacteria mainly belonging to Leuconostoc, Lactobacillus and Streptococcus genera and therefore its molecular weight and branching is dependent on the bacteria strand, pH of the medium and concentration of sucrose in the medium.^[53,54]

Figure 15: Possible Structure of Dextran.

As a bioproduct, dextran is biocompatible and -degradable and thus has many applications in the food and pharmaceutical industry. The biodegradation is possible through the enzyme dextranase, which can be produced by bacteria.^[53] Dextran has a wide range of applications which are derived from the different physicochemical properties of dextran of different molecular weights and degrees of branching. Intrinsic bioactivity of dextran can be shown by dextran sulphate with an antiviral effect against human immunodeficiency virus and dextran as potential antiviral- and immunomodulatory agents in trout.^[53] Due to its biocompatibility, nontoxicity and facile chemical modification, crosslinking dextran chains can lead to semi-synthetic hydrogels for tissue engineering. Cross-linking dextran with dithiothreitol yields a hydrogel, which can encapsulate rat bone marrow mesenchymal stem cells.^[43]

20

1.3 Purification and characterization methods

1.3.1 Reversed-phase high performance liquid chromatography (RP-HPLC) and size-exclusion chromatography (SEC)

Several similar chromatography techniques are used for the separation of different molecular species. The molecular species of interest (analyte) must be separated from the matrix (rest of components) in the sample. Therefore, chromatography is used to separate the analyte from the sample by introducing it to a flowing mobile phase that must pass over a stationary phase. The stationary phase shows varying interaction strength with different kinds of molecular species in the sample and thus releases them separately back into the mobile phase.

The mobile phase can be either a gas (gas chromatography) or a liquid (liquid chromatography), but it is also possible to have a supercritical fluid, countercurrent or electrochromatography. Focusing on the HPLC, the stationary phase is usually a column packed with porous particles having a diameter ranging from 1-5 µm and the mobile phase (eluent) a solvent that moves through the column by a pump at elevated pressure. The exiting molecular species can be detected by different methods, depending on the physicochemical properties of the analyte. The most commonly used methods for detection use ultraviolet(UV)-absorption, refraction index, fluorescence, molecular mass or fragmentation in a mass spectrometer.^[55]

21 Figure 16: Schematic representation of a simple HPLC system. 1) Solvent supply system with a solvent container and degasser, 2) pumping system for high pressure, 3) injector (syringe) with the sample and switching valve for A loading the loop and B injecting the sample, 4) chromatographic column with a possible precolumn, 5) one or more possible detectors, 6) controller/ data processing unit.^[55]

Figure 16 shows a possible set-up for HPLC instrument. The solvent supply system consists of one or more reservoirs and a possible degasser, to remove gases dissolved in the eluent. The pumping system delivers constant flow of solvent via one or more pumps through the complete HPLC system. They must be able to produce and maintain high pressure, to overcome the resistance to flow of the chromatographic column. This flow is characterized by the volumetric flow rate U, which can take up values between 0.1 to 10 mL/min with a pressure between 6000 to 8500 psi. A small, precisely measured volume of a solution containing the sample can be added to the mobile phase through the injector. One method to inject a sample in the system is the loop system pictured in Figure 16. The loop can be filled with the sample and afterward connected to the flow circuit by switching a valve. Depending on the type of HPLC setup that is used, the size of the loop can vary between 20 nL to some milliliters. The role of chromatographic columns in HPLC is to achieve the separation of the analyte. Columns usually consist of a tube made from metal (stainless steel) or plastic (e.g.,

22

polyether ketone) and is filled with the stationary phase, which is held in place by two special frits at the ends of the tube. The dimensions of the tube vary depending on the use. The length of a typical chromatographic column can be between 30 to 250 mm and have an internal diameter between 1 to 10 mm. This tube can be filled with different kinds of stationary phases depending on the separation method: Normal phase, reversed phase, ion exchange size exclusion, etc. The stationary phase consists of small, solid particles with different porosity (porous, superficially porous and pellicular) and surface properties. Porous particles have a diameter of 3 to 5 µm and consist of specific porous materials like silica with a surface coating of active components. Those surfactants are physically or chemically bound to the surface and are the components interacting with the mobile phase. The chemical properties of the surface of the particles can be altered by changing the chemical nature of the active surface, chemical stability, surface reactivity or density and distribution of the reactive centers. RP-HPLC for example uses octadecyl (C18) or octyl (C8) groups on the surface of silica. Size-exclusion chromatography uses perfusion particles made from different kinds of polymers or silica with big pores (400-800 nm) connected to a system of small pores (30-100 nm). In some systems, there is more than one column required, to get the desired separation, thus two to four columns are connected in series. Also, there is the option or requirement to heat a column to reach the preferred separation. Therefore, the column is put in a column oven, which can control the temperature between 10-100 °C.^[55]

1.3.2 Matrix-assisted laser desorption/ionization-time of flight mass spectroscopy (MALDI-ToF MS)

Besides electrospray ionization, MALDI is one of two soft ionization techniques and allows the detection of large, non-volatile and labile molecules by mass spectrometry. This technique evolved from a diversity of different methods developed over time. In the 1960s Beckey introduced field desorption, which opened the possibility to analyze bioorganic molecules via MS. A step further in the direction of MALDI-ToF MS was taken, when the secondary ion MS introduced by Benninghoven 1975 was combined by Barber 1981 with glycerol as “matrix”, to promote desorption and enhance ion yield. The invention of the laser enabled the generation of ions for MS analysis via laser irradiation. Over several years, MALDI was further improved

23 to measure samples with higher molecular masses like proteins (10-100 kDa) and promote the sensitivity of the detector, which can nowadays detect ions in the attomolar range.^[56]

Figure 17: Basic principle of MALDI-ToF MS.^[57]

The basic principle of MALDI-ToF MS is the co-crystallization of the molecule of interest with a matrix and ionization of the mixture with a laser. This matrix is used in excess of the sample and enables the efficient absorption of the laser energy at the operated wavelength. The sample desorbs and “gently” ionizes together with the matrix through the energy of the laser, to prevent the fragmentation of the labile sample. After the acceleration through an electric field, the ions are directed across an evacuated flight tube to a detector, which detects the series of impacts of the ions. Therefore, the mass-to-charge ratio of the analyzed molecules can be determined, by knowing the time-of-flight to impact on the detector. The detectors can run in positive or negative ion mode and there are different options for the matrix like α-cyano-4-hydroxycinamic acid (HCCA) and 2,4-dihydroxybenzoic acid (DHBA) and their derivates. Figure 17 shows the basic principle of MALDI-ToF MS from the desorption/ionization of the sample to detection and visualizing the data.^[57]

1.3.3 Thioflavin T-Assay

The aggregation of peptide/proteins to form amyloid fibrils can cause a wide range of human disorders like Alzheimer’s disease, Parkinsons’s disease and type 2 diabetes. Thioflavin T (ThT) is a commonly used dye to monitor amyloid fibril formation. Bound to an β-sheet rich fibril,

24

ThT exhibits a strong fluorescence signal at approximately 482 nm upon excitation at approximately 450 nm. The fluorescence is enhanced through rotational immobilization of the central C-C bond connecting the benzothiazole and the aniline rings upon binding to an β-sheet fibril. ThT binds to the side chain channels (“channel” model) of the fibrils formed by four or more consecutive β-strands along the long axis of the amyloids (Figure 18).^[58,59]

Besides amyloid fibrils, the C-C bond rotation can be suppressed by cyclodextrin, cucurbit[n]uril, polymer membranes, porous silicon and other biomolecules like DNA, which yields in this context to falsely positive results.^[60] ThT binds to α-helices with Tyr- and Trp-rich areas due to π-stacking or to hydrophobic pockets of human serum albumin or drug-like molecules.^[61]

Figure 18: Chemical structure and spatial model of ThT cation (left). Benzthiazole ring (I), benzene ring (II), and dimethylamino group (III) are boxed.^[59] Cross-β structure of amyloid fibrils, formed from layers of laminated β-sheets and “Channel” model of ThT binding to fibril-like β-sheets (right).^[61]

25 1.3.4 Conversion-Assay

The conversion of molecularly dissolved peptides to peptide aggregates/β-sheet fibrils is determined by a conversion-assay. For this purpose, an incubated peptide solution is passed through a spin tube with a certain molecular weight cut-off (MWCO) via centrifugation.

Aggregates and β-sheet fibrils are too large to pass through the filter, thus only single molecules are collected. As a reference, the same amount of peptide solution is used without filtration (original). Both solutions are lyophilized and re-dissolved in dimethyl sulfoxide (DMSO) to mix with fluorescamine.^[8] As a fluorescence marker, fluorescamine reacts with primary amines, like peptides, to exhibit fluorescence (Figure 19).^[62]

Figure 19: Reaction of fluorescamine with primary amines to form a fluorophore.^[62]

Upon excitation (𝜆_exc = 365 nm), the fluorophore emits light with the emission wavelength 𝜆_em= 470 nm. The fluorescence intensity of the original solution 𝐼_Original reflects the whole number of peptides, whereas the fluorescence intensity of the filtrate 𝐼_Filtrate reflects only the non-fibrillated peptides. The conversion rate 𝐶𝑅 can be calculated with the following formula:

𝐶𝑅 = 100 −100 ∗ 𝐼_Filtrate

𝐼_Original [%] (1)^[8]

1.3.5 Transmission electron microscopy (TEM)

A microscope is used to magnify objects too small to see with the naked eye. The smallest distance between two points our eyes can resolve is approximately 0.1 mm and based on the wavelength 𝜆 and numerical aperture 𝑁𝐴. The numerical aperture consists of the product of the refractive index 𝜇 and the sinus of angular aperture sin 𝛽. This smallest distance between two points that can be resolved is called Abbe limit 𝛿 and can be calculated via the Rayleigh criterion:^[63]

26

𝛿 =0.61𝜆

𝑁𝐴 (2)^[63]

In most cases the sample is measured in air or vacuum, thus the refractive index 𝜇 equals 1.

The sinus of the angular aperture sin 𝛽 is usually nearly 1, thus the whole numerical aperture 𝑁𝐴 can be simplified to 1. With this simplification, the Abbe limit 𝛿 is only dependent on the wavelength of the radiated light of the light source. In the case of visible-light microscopes, the highest resolution possible is around 300 nm (green light: 𝜆 = 550 nm). To overcome those limits, electrons are used instead of photons, which wavelength is dependent on its energy 𝐸.Ignoring the relativistic effects, the wavelength of an electron can be calculated with the following formula:

𝜆 =1.22 𝐸¹²

(3)^[63]

The wavelength 𝜆 for an electron with energy 𝐸 = 100 keV equals 4 pm and thus the Abbe limit 𝛿 = 2.4 pm. However, it is not possible to build a perfect TEM because of limits of the electron lenses, thus such high resolutions cannot be reached. The high resolution of TEM imaging brings some limitation with it. It is only possible to look at small part of the sample at any time. Thus, it is important to analyze the sample with other microscopy methods or the eye before looking at small parts of the specimen. Also, TEM imaging presents only 2D images of 3D samples. Therefore, the interpretation of the image is key, before making false conclusions. A further problem is the damage to the sample through ionizing radiation.

Particularly polymers and biological specimens are easily destroyed by an electron beam.

However, the combination of intense electron beams with sensitive electron detectors and by using computer enhanced noisy images, the total dose of electrons received by the sample can be reduced below the damage threshold. The last limitation of TEM imaging is the thickness of the sample. The specimen must be thin enough for electrons to pass through the sample (electron transparency) and thus should be <100 nm. In the case of high resolution TEM imaging the sample must be 50 nm or even thinner.^[63]

Upon impact the electrons produce a wide variety of secondary signals, which can be detected by different TEM methods (Figure 20). Most of those signals are used in analytical electron microscopy to get chemical information and further details of the sample. For TEM imaging especially the direct beam is important to visualize the investigated specimen.^[63]

27 Figure 20: Generated signals by interaction of high-energy beam of electrons with a thin specimen.^[63]

The simplest setting for a TEM consist of the electron source, three different lenses and their apertures and the detector. There are two different types of electron sources: thermionic and field-emission sources. The thermionic source consists of either tungsten filaments or lanthanum hexaboride crystals and produce electrons by heating. Field emitters are fine tungsten needles, which produce electrons when large electric potentials are applied between them and an anode. The electron lenses are the TEM’s equivalent of glass lenses for visible light microscopy and can be discussed in similar fashion. Lenses, in principle, have two basic functions: 1. Collects all beams radiated from a point in an object and reconstruct a point in an image; 2. Focus parallel beams to a point in the focal plane of the lens. The objective lens is the most important lens in the TEM and forms the images and diffraction pattern that are magnified by the other lenses. The intermediate lens selects either the back-focal plane for the diffraction or the image plane for the image. The projector lens focuses the final image or diffraction pattern on the detector or viewing screen. The condenser, objective and intermediate aperture select only the relevant electron beam after the corresponding lens.

Figure 21 shows the two basic operation modes of a TEM imaging system. Depending on the selected aperture, the diffraction or image is projected on the screen. Since the imaging mode is relevant for viewing self-assembled peptides or cross-linked polymer backbones, the diffraction mode is not further explained. The image mode can be differentiated into bright- and dark-field imaging. In the case of the bright-field, only the direct beam is selected to form the bright-field image. The dark-field shows only electrons, that are not in the direct beam.

28

Figure 21: The two basic operations of the TEM imaging system: diffraction (left) and imaging mode (right). Diffraction mode projects the diffraction pattern onto the viewing screen with the intermediate lens selecting the back-focal plane. Imaging mode projects the image onto the viewing screen with the image lens selected (Note: highly simplified diagram).^[63]

1.3.6 Attenuated total reflection-Fourier transformation infrared spectroscopy (ATR-FTIR)

IR spectroscopy is utilized to analyze the amide bond of peptides and proteins. The frequencies, at which the amide bond vibrations occur, can be assigned to different secondary structures of the peptide/protein. Especially the Amide I, II and III IR spectral regions can be used for protein structure analysis. Due to the variance of hydrogen bonding among the AA, different vibrations of the amide bonds occur. For example, α-helices and β-sheets have different folded structures and while they both form highly ordered structures, their signal patterns are distinct. This difference in hydrogen bonding and the geometric orientation of the amide bonds in the corresponding structure gives rise to different vibration frequencies associated with the individual secondary structures. The amide bond involves the vibrations

29 of three different groups: C=O, C-N and N-H. Those vibrations can be assigned to three major spectral regions, as mentioned before. The amide I vibration region is located between 1700-1600 cm^-1 and is widely used due to its strong signal. This region is corresponding to the C=O stretch, C-N stretch and N-H bending. The amide II region involves the frequencies between 1600-1500 cm^-1 and represents the C-N stretch and the N-H bending. At last, the amide III region represents the N-H in plane bending and the C-N stretching from 1350-1200 cm^-1. It also includes C-H and N-H deformation vibration.^[64]

The amide I vibration region is mainly used to characterize the secondary structure of peptides and proteins. Therefore, frequencies were determined, which correlate to the secondary structures in proteins. The α-helical structure shows bands around 1661-1647.5 cm^-1. Furthermore, the β-sheet structure shows bands around 1689-1682 cm^-1, 1637.5-1627.5 cm^-1 and 1627.5-1615 cm^-1. Unordered or random structures can be assigned to the bands between 1644.5-1637.5 cm^-1 and finally β-turns can be assigned to frequencies between 1682-1661 cm^-1.^[65]

Due to the O-H vibrations of water (1640 cm^-1), it is difficult to measure aqueous peptide/protein solutions.^[66] Therefore, peptide/protein solutions can be lyophilized to receive dry powders. Those can be easily measured in low quantities with precise results.^[8]

The ATR-unit makes use of the high refractive index of a dielectric, which totally reflects radiation at an angle larger than the critical angle, if a sample is introduced to the surface. The reflection will not be total anymore at frequencies, where the sample absorbs the radiation, thus a reflection spectrum with high contrast and intensity is obtained, which resembles a transmission spectrum. Therefore, if the sample is non-absorbing, the incident beam is reflected without energy loss. However, if the sample absorbs energy, a periodic alternation takes place and the reflection is not total anymore.^[67]

The ATR-unit makes use of the high refractive index of a dielectric, which totally reflects radiation at an angle larger than the critical angle, if a sample is introduced to the surface. The reflection will not be total anymore at frequencies, where the sample absorbs the radiation, thus a reflection spectrum with high contrast and intensity is obtained, which resembles a transmission spectrum. Therefore, if the sample is non-absorbing, the incident beam is reflected without energy loss. However, if the sample absorbs energy, a periodic alternation takes place and the reflection is not total anymore.^[67]